Distribution of Neuronal ApoptosisInhibitory Protein-Like Immunoreactivity

in the Rat Central Nervous System

D.G. XU,1 R.G. KORNELUK,2 K. TAMAI,3 N. WIGLE,1 A. HAKIM,4

A. MACKENZIE,2 AND G.S. ROBERTSON1*1Department of Pharmacology, Faculty of Medicine, University of Ottawa,

Ottawa, Ontario K1H 8M5, Canada2Molecular Genetics Research Institute, Children’s Hospital of Eastern Ontario,

Ottawa, Ontario K1H 8L1, Canada3Medical and Biological Laboratories Co., Ltd., Naka-ku, Nagoya 460, Japan4Neuroscience Research Institute, Faculty of Medicine, University of Ottawa,

Ottawa, Ontario K1H 8M5, Canada

ABSTRACTWe have recently shown that spinal muscular atrophy (SMA), an autosomal recessive

disorder characterized by motor neuron loss, is associated with deletion of a gene that encodesthe neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined thedistribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system(CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain,immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells,dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basalforebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateraland ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. Inthe midbrain, NAIP-LI was located primarily within neurons of the red nucleus, thesubstantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In thebrainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal,facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine,olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum,NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei).Finally, within the spinal cord, NAIP-LI was detected in Clarke’s column and in motorneurons. Taken together, these results indicate that NAIP-LI is distributed broadly in theCNS. However, high levels of NAIP-LI were restricted to those neuronal populations that havebeen reported to degenerate in SMA. This anatomical correspondence provides additionalevidence for NAIP involvement in the neurodegeneration observed in acute SMA. J. Comp.Neuro. 382:247–259, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: neuronal apoptosis inhibitory protein; apoptosis; cranial nerve; motor neuron;

spinal muscular atrophy

Developmental cell death, or apoptosis, is a naturallyoccurring process that is thought to play a critical role inestablishing appropriate neuronal connections in the devel-oping central nervous system (CNS; Johnson and Deck-werth, 1993). Apoptosis is characterized morphologically bycondensation of the chromatin followed by shrinkage of thecell body. Biochemically, the hallmark of apoptosis is thedegradation of nuclear DNA into oligonucleosomal frag-ments (multiples of 180 base pairs) mediated by a Ca21/Mg21-dependent endonuclease. DNA laddering precedescell death and may be a key event leading to death. In

keeping with this proposal, agents that inhibit DNAfragmentation prevent apoptosis, whereas morphologyindicative of apoptosis is produced by enzymes that digest

Contract grant sponsor: Heart and Stroke Foundation of Ontario;Contract grant number: NA-2938; Contract grant sponsor: London Life.*Correspondence to: G.S. Robertson, Department of Pharmacology, Fac-

ulty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5,Canada. E-mail: grobert@labsun1.med.uottawa.caReceived 28 October 1996; Revised 18 February 1997; Accepted 18

February 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 382:247–259 (1997)

r 1997 WILEY-LISS, INC.

nuclear DNA (Appleby and Modak, 1977; Arends et al.,1990). Apoptosis is often dependent on RNA and proteinsynthesis within the dying cell, suggesting the activationof a cell-death pathway. The best-defined genetic pathwayof cell death is in the nematode Caenorhabditis, whereboth effector (ced-3 and ced-4) and repressor (ced-9) geneshave been isolated (Yuan andHorvitz, 1990, 1992). Similargenes have been identified inmammals. One such exampleis the protooncogene bcl-2, which is thought to be themammalian homolog of ced-9 (Tsujimoto et al., 1986;Bakhshi et al., 1985; Cleary et al., 1986). Overexpressionof Bcl-2 has been shown to render neurons resistant to the

damaging effects of a wide variety of noxious treatments(for review, see Reed, 1994). However, the very low levels ofBcl-2 detected in adult brain suggest that other proteins

Abbreviations

3 oculomotor nucleus3nr oculomotor nerve root7 facial nucleus8vn vestibulocochlear nerve10 nucleus vagus12 hypoglossal nucleusC5 cervical 5CC Clarke’s columnCu cuneate nucleusDA dorsal hypothalamic areaDG dentate gyrusDMSP5 dorsal medial spinal trigeminal nucleusEP endopeduncular nucleusEW Edinger-Westphal nucleusG granule cell layerGP globus pallidusGr gracile nucleusHDB horizontal diagonal bandI–VI cortical layersint interposed cerebellar nucleusL stratum lucunosumlat lateral cerebellar nucleusLC locus coeruleusLH lateral hypothalamic area

LHb lateral habenular nucleiM stratummoleculareMe5 mesencephalic trigeminal nucleusMHb medial habenuclar nucleusMo5 motor trigeminal nucleusO stratum oriensP pyramidal cell layer (Fig. 2), Purkinje cell layer (Fig. 11)Pn pontine nucleusPr5 principal trigeminal sensory nucleusR stratum radiatumRMC red nucleus, magnocellular partRPC red nucleus, parvocellular partRPO rostral periolivary areaS sacral spinal cordSNC substantia nigra pars compactaSNL substantia nigra pars lateralisSNR substantia nigra pars reticulataSP5O spinal trigeminal nucleus, oral partT2 thoracic 2T6 thoracic 6VC ventral cochlear nucleusVDB vertical diagonal bandVe vestibular nucleus

Fig. 1. Western blotting analysis of antisera for neuronal apoptosisinhibitory protein (NAIP). Nitrocellulose stripes electrophoreticallytransferred with whole cell extracts from rat brains were blotted witheither NAIP antisera or NAIP antisera that had been preabsorbedwith glutathione-S-transferase (GST)-NAIP fusion protein. The affinity-purifiedNAIP antibody recognized a single band at 150 kD correspond-ing to the predicted molecular weight for NAIP (lane A). Preabsorp-tion of the antibody with GST-NAIP fusion protein eliminated the 150kD band (lane B). Each lane was loaded with 20 µg of protein.

Fig. 2. NAIP-like immunoreactivity (NAIP-LI) in the neocortex. A:Neuronal perikarya and processes were moderately labelled in allcortical layers I–VI. B: NAIP-LI in a layer V pyramidal neuron. Notethe intense labelling in the apical dendrite (arrowhead). Scale bars 5300 µm inA, 30 µm in B.

248 D.G. XU ET AL.

may play a more important role in preventing apoptosis inthe mature CNS (Krajewski et al., 1995).Spinal muscular atrophy (SMA) is a hereditary neurode-

generative disorder that is characterized by a severedepletion of motor neurons in the spinal cord and brain-stem (Byers and Banker, 1961). Many of the motor neu-rons observed at autopsy in SMA spinal cords displayfeatures, such as chromatolysis, that are consistent withapoptosis (Murayama et al., 1991). During maturation ofthe spinal cord, as much as 50% of motor neurons undergoapoptosis. This has led to the suggestion that a geneticdefect in a neuronal apoptotic pathway may be responsiblefor motor neuron depletion in SMA (Sarnat, 1983). Re-cently, two candidate genes, i.e., survival motor neuron(smn; Lefebvre et al., 1995) and neuronal apoptosis inhibi-tory protein (naip; Roy et al., 1995), have been identified.Acute SMA is associated with deletion of naip (Roy et al.,1995). The product of this gene was termed neuronalapoptotic inhibitory protein (NAIP) because of sequencehomology with two baculoviral proteins (Cp-IAP and Op-IAP) that block virally induced apoptosis, and, recently, ithas been shown to inhibit apoptosis induced by a variety oftriggers in several different cell lines (Roy et al., 1995;Liston et al., 1996) and by global ischemia in the rat (Xu etal., unpublished observations).In acute forms of SMA, neuropathological involvement

may extend well beyond the lower motor neuron territoryof the spinal cord to include a wide variety of structuresextending from the hindbrain to the forebrain (Steiman etal., 1980; Towfighi et al., 1985). At present, it is not clearwhether degeneration in supraspinal sites is indicative ofSMA or of epiphenomenon resulting from such factors ashypoxia or nutritional deficiency. Given the possibility thatdepletion of NAIP may be a major contributing factor inSMA, we sought to determine whether NAIP was presentin those neuronal populations that are at risk in SMA.This was accomplished by immunohistochemistry usingan affinity-purified polyclonal antibody against NAIP. Weshow that, in the adult rat CNS, there is an excellentcorrespondence between neurons that display NAIP-likeimmunoreactivity (NAIP-LI) and those neuronal popula-tions that have been reported to suffer pathological alter-ations in severe forms of SMA.

MATERIALS AND METHODS

Animals

Adult male Wistar rats (250–275 g; Charles Rivers,Montreal, Canada) were housed two per cage in a tempera-

TABLE 1. Distribution of Neuronal Apoptosis Inhibitory ProteinImmunoreactivity in the Rat Central Nervous System1

Areas examined NAIP-LI

TelencephalonCortexNeocortex 1Piriform 1Perirhinal 1Entorhinal 1Cingulate 1

HippocampusPrincipal neuronsPyramidal cells 1Granule cells 1

Interneurons 1Basal gangliaCaudate putamen 2

Medium-sized neurons 1Cholinergic interneurons 11

Globus pallidus 112

Ventral pallidum 11Endopeduncular nu. 11

Amygdala 1DiencephalonHabenulaMedial habenular nu. 1Lateral habenular nu. 11

ThalamusParaventricular thal. nu. 1Intermediodorsal thal. nu. 1Mediodorsal thal. nu. 1Central medial thal. nu. 1Rhomboid thal. nu. 1Reuniens thal. nu. 1Centrolateral thal. nu. 1Paracentral thal. nu. 1Gelatinosus thal. nu. 1Laterodorsal thal. nu. 1Posterior thal. nu. group 1Ventroposterior thal. nu. 112

Ventromedial thal. nu. 112

Ventrolateral thal. nu. 112

Reticular thal. nu. 1HypothalamusDorsomedial hypothal. nu. 1Ventromedial hypothal. nu. 1Dorsal hypothal. area 1 (Scattered)Lateral hypothal. area 1 (Scattered)Arcuate hypothal. nu. 1

MesencephalonSubstantia nigra 2

Pars compacta 11Pars reticulata 1Pars lateralis 1

Red nu. 112

Ventral tegmental area 1Periaqueductal gray 1Interpeduncular nu. 11

Met- and mylencephalonCranial nerve nucleusOculomotor 112

Trochlear 112

Abducens 1Trigeminal sensory complex 2

Main sensory trigeminal 11Spinal sensory trigeminal 11

Trigeminal motor 112

Facial 112

Cochlear 112

Ambiguus 112

Vagus 112

Hypoglossal 112

Vestibular 112

Solitaris 112

Brainstem relay and reticular nucleusCuneate 112

Gracile 112

Inferior olivary 112

Lateral reticular 112

Pontine tegmental reticular formation 1Pontine 112

Raphe 1Locus coeruleus 112

CerebellumCerebellar cortexPurkinje cells 112

Granule cells 2Cerebellar nucleusLateral 112

Interposed 112

TABLE 1. (continued).

Areas examined NAIP-LI

Met- and mylencephalon (continued)Spinal cordLamina IX 112

Lamina VIII 112

Lamina VII 112

Lamina VI 1Laminae I–V 1

1The intensity of neuronal apoptosis inhibitory protein-like immunoreactivity (NAIP-LI) in the examined areas was categorized either high or low by comparison with theintensity observed in facial neurons that were intensely labelled. Immunoreactivity thatappeared to be equivalent to that seen in this neuronal population was categorized ashigh (11). Less intense labelling was classified as low (1). Neuronal populations thatfailed to display labelling are indicated by (2).2Regions in which damage has been observed in cases of acute spinal muscular atrophy(SMA; Steiman et al., 1980; Towfighi et al., 1985; Murayama et al., 1991).

NAIP-LI IN THE CNS 249

ture-controlled environment with a 12 hour light/12 hourdark cycle and were given free access to water and Purinalaboratory chow. All animal procedures conformed to theGuide to the Care and Use of Experimental Animalsendorsed by the Medical Research Council of Canada.

Experimental protocols

In a first experiment, the specificity of NAIP antiserawas assessed by both immunohistochemistry and Westernblotting. In this study, we determined whether preabsorp-tion of the antisera with glutathione-S-transferase (GST)-NAIP fusion protein eliminated NAIP-LI. In a secondexperiment, immunohistochemical localization of NAIP-LIwas examined in coronal sections cut from a variety oflevels in the forebrain, midbrain, hindbrain, and spinalcord. In a third experiment, double-immunofluorescencelabelling was performed by using antibodies against NAIPand several marker proteins, which included choline acet-yltransferase (ChAT), tyrosine hydroxylase (TH), and cal-bindin D-28K (CaBP), to examine the localization ofNAIP-LI in cholinergic, dopaminergic, and Purkinje neu-rons, respectively.

Tissue preparation

Animals were anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 200 ml of saline

(0.9%) followed by 150 ml of phosphate buffer (0.1 M)containing 4% paraformaldehyde. Brains were postfixedovernight and cryoprotected in phosphate buffer (0.01 M)containing 10% sucrose for 2 days. Sections (12 µm thick)were cut from the appropriate brain region by using acryostat and were processed for NAIP-LI.

Antibodies

An affinity-purified polyclonal rabbit antibody was usedto detect NAIP. This antibody was generated in thefollowing manner. NAIP cDNA cd23.2 (Roy et al., 1995)was amplified by polymerase chain reaction (PCR) usinglambda gt11 primers and was digested with EcoRI (Gibco,Gaithersburg, MD), and a 1 kb fragment encoding exons7–11 was subcloned into the EcoRI site of the fusion vectorpGEX-3X. Rabbits were immunized with purified, bacteri-ally produced fusion protein in complete Freund’s adju-vant. Serum was precleared with GST protein and anti-NAIP immunoglobulin purified with immobilized GST-NAIPfusion proteins (constructed by Dr. Alex Mackenzie byusing a Pharmacia fusion protein plasmid; pGEX-4T-3).The antibody used for detection of ChAT-like immunoreac-tivity (ChAT-LI) was an affinity-purified goat antibodyraised against human placenta ChAT (Chemicon Interna-tional Inc., Temecula, CA). Dopaminergic neurons in themidbrain were identified with a monoclonal antibody

Fig. 3. NAIP-LI in the hippocampus. NAIP-LI was detected inpyramidal neurons (CA1–CA3), dentate granule cells (DG), andinterneurons (A). Within the CA1 subfield, NAIP-LI was present inthe cell bodies and dendrites of pyramidal neurons (B). The intensityof NAIP-LI exhibited by neurons in subfields CA1–CA3 and thedentate gyrus appeared similar (B–D). Compared with these neuronal

populations, more intense staining was apparent in interneuronsscattered throughout the hippocampus in stratum oriens, lucunosumof CA1 (B), lucidum of CA2 and CA3 (C), and the dentate hilus (D).Arrowheads indicate interneurons. Scale bars 5 500 µm in A, 300 µmin B–D.

250 D.G. XU ET AL.

Fig. 4. NAIP-LI in the striatum and double-labelling immunohisto-fluorescence of striatal neurons for NAIP and choline acetyltransfer-ase (ChAT). A: Large neurons that displayed intense labelling wereobserved throughout the caudate putamen (arrowhead). By contrast,medium-sized neurons were weakly labelled with the NAIP antibody(arrow). B,C: Double-labelling immunofluorescence showing a perfectoverlap between NAIP-LI (B; arrowheads) and ChAT-immunoreactiveneurons (C; arrowheads). Scale bars 5 300 µm inA, 30 µm in B,C.

Fig. 5. NAIP-LI in output structures of the basal ganglia. Labelledneurons were observed in the globus pallidus (GP;A), endopeduncularnucleus (EP; B), and substantia nigra (SNL, SNC, SNR; C). In thesestructures, especially the interpeduncular nucleus, strongly labelledfibers were also seen (B). In the substantia nigra, NAIP1 neurons werelocated predominantly in the substantia nigra pars compacta (SNC).Scattered immunoreactive neurons were also present in the substan-tia nigra pars reticulata (SNR), and the substantia nigra pars lateralis(SNL). Dendrites originating from the pars compacta were observedextending ventrally into the SNR (C). Scale bars 5 300 µm.

NAIP-LI IN THE CNS 251

against TH (Incstar, Stillwater, MN), whereas a monoclo-nal antibody against CaBP (Sigma, St. Louis, MO) wasused to localize Purkinje cells in the cerebellar cortex.

Immunohistochemistry

Immunohistochemistry was performed by using a stan-dard procedure described previously by Robertson andFibiger (1992). Briefly, sections (12 µm thick) were washedin 0.02 M phosphate-buffered saline (PBS) containing0.3% hydrogen peroxide for 10 minutes to block endog-enous peroxidase activity. Sections were then washedthree times in PBS and were incubated in PBS containing0.3% Triton X-100, 0.02% sodium azide, and NAIP (diluted1:750) primary antisera for 48 hours. Next, the sectionswere washed three times with PBS and were incubatedwith biotin-labelled donkey anti-rabbit secondary antisera(1:500) for 16 hours. The sections were then washed threetimes with PBS and were incubated for 3 hours with PBScontaining 0.3% Triton X-100 and streptavidin-horserad-ish peroxidase (1:200; Amersham, Arlington Heights, IL).After three washes in PBS, the sections were rinsed in 0.1M acetate buffer, pH 6.0. The reaction was visualized byusing a glucose oxidase-diaminobenzidine (DAB)-nickelmethod described previously (Shu et al., 1988). The reac-tion was terminated by washing in acetate buffer, andsections were mounted on chrom-alum-coated slides. Afterdrying, the sections were dehydrated through a gradedseries of alcohols and two changes of xylene, and they werecoverslipped for microscopic observation.

Double-labelling immunohistofluorescence

Sections from the striatum, basal forebrain, brainstem,and spinal cord were incubated with rabbit anti-NAIP(1:300) and anti-ChAT (1:500), ventral mesencephalic sec-tions were incubated with anti-NAIP (1:300) and anti-TH(1:500), and cerebellar sections were incubated with anti-NAIP (1:300) and anti-CaBP (1:500) for 48 hours at 4°C inPBS containing 0.3% Triton X-100. Sections were rinsed inPBS and were then incubated with a mixture of indocarbo-cyanine (Cy3)-labelled donkey anti-rabbit and the appropri-ate biotinylated secondary antisera (1:100 and 1:50; Amer-sham) prepared 24 hours prior to use. After 2 hours atroom temperature and 1 hour at 37°C, sections were rinsedin PBS. Sections were then incubated for 3 hours at roomtemperature with streptavidin-fluoroisothiocyanate (FITC;1:50; Amersham) in PBS containing Triton X-100. Slideswere coverslipped by using a mounting medium of PBScontaining phenylaminediamine (0.1 mM) and 90% glyc-erol and were examined by using a Zeiss Axioplan micro-scope equipped with both single- and dual-filter sets forthe visualization of FITC and Cy3.

RESULTS

The affinity-purified NAIP antibody recognized a singleband at 150 kD, which corresponds to the predictedmolecular weight for NAIP (Fig. 1). In contrast, antiserathat had been preabsorbed with NAIP fusion protein failedto detect this band. Neuronal labelling was detected byimmunohistochemistry with the affinity-purified antibody.This labelling was eliminated by preabsorption of theantisera with GST-NAIP fusion protein (data not shown).These results indicate that this antibody selectively recog-nizes NAIP.

Telencephalon

Cortex. Neurons that displayed NAIP-LI were ob-served in all layers of the neocortex (Fig. 2A, II–VI).

Fig. 6. NAIP-LI in the diagonal band and double-labelling immuno-histofluorescence of diagonal band neurons for NAIP and ChAT.NAIP-immunoreactive neurons were present in the horizontal (HDB)as well as the vertical (VDB) limb of the diagonal band (A). Doublelabelling revealed little overlap between NAIP1 (B) and ChAT1

neurons (C) in the diagonal band. Arrowheads indicate a singledouble-labelled neuron. Scale bars 5 300 µm in A, 30 µm in C (alsoapplies to B).

252 D.G. XU ET AL.

Low-to-moderate levels of NAIP-LI were detected in allcortical regions examined (Table 1). Immunoreactive label-ling in the cell body was moderate and was confined to thecytoplasm. By comparison, dendritic staining was moreintense, with the most prominent labelling observed in theapical dendrites of layer V pyramidal neurons (Fig. 2B).Hippocampus. In the hippocampus, all of the pyrami-

dal neurons in the CA1–CA3 subfields as well as granulecells in the dentate gyrus displayed moderate levels ofimmunoreactivity that were apparent in both perikaryaand processes (Fig. 3A–C). Interneurons scattered through-out the hippocampus in the oriens layer, stratum radia-tum, and lucunosum moleculare of CA1 as well as thestratum lucidum of CA2 and CA3 displayed intense label-ling (Fig. 3B–D).Striatum. Strongly labelled neurons were observed in

the caudate putamen and the nucleus accumbens. Theseimmunoreactive neurons were large (25–30 µm) and repre-sented a very small proportion, approximately 1%, of thetotal striatal neuron population (Fig. 4A). Consistent withthese findings, double labelling with ChAT antisera re-vealed that NAIP-LI was localized exclusively in choliner-gic interneurons (Fig. 4B,C). All of the other neurons inthis structure exhibited very low levels of NAIP-LI.

Globus pallidus. Large, intensely stained bipolar neu-rons were observed in this striatal projection site (Fig. 5A).Basal forebrain. Moderately immunoreactive neu-

rons were detected in both the horizontal and the ventrallimbs of the diagonal band of Broca as well as in themedialseptal nucleus and the ventral pallidum (Fig. 6A, Table 1).The distribution of these NAIP-immunoreactive cell bod-ies appeared to match the distribution of cholinergicneurons in the basal forebrain. However, double labellingfor NAIP and ChAT revealed little colocalization of NAIP-and ChAT-LI in the medial septum and the diagonal band(Fig. 6B,C).

Diencephalon

Thalamus. Moderate-to-high intensity labelling wasobserved in a large number of medium-sized neuronssituated in ventral and lateral portions of the thalamus(Fig. 7A,B).Although they were fewer in number, immuno-reactive neurons were also detected in anterior, intralami-nar, and medial aspects of the thalamus (Table 1).Habenula. Numerous small, round,moderately immu-

noreactive perikarya were seen in the medial habenularnucleus. In the lateral habenular nucleus, a few intenselystained neurons of medium size were detected (Fig. 7C).

Fig. 7. NAIP-LI in the diencephalon. A: NAIP-LI was detected innearly all thalamic areas, with more intensely labelled neuronsapparent in the ventromedial and ventral posterior nuclei. B: Highermagnification of the squared area indicated in A showing labelledneurons in the ventral posterior nucleus. C: In the habenula, NAIP-LI

was displayed by both medial (MHb) and lateral (LHb) habenularnuclei. A small population of large bipolar neurons in the lateralhebenula exhibited very strong NAIP-LI. D: In the hypothalamus,NAIP-LI was rarely detected. Scale bars 5 600 µm in A, 300 µmin C,D.

NAIP-LI IN THE CNS 253

Hypothalamus. Compared with the thalamus, littleNAIP-LI was seen in the hypothalamus.Asmall number ofweakly stained neuronswas detected in the arcuate nucleusas well as in dorsal and lateral hypothalamic areas (Fig.7D, Table 1).Entopeduncular nucleus. Intense labelling was ob-

served in this basal ganglia output. In addition to peri-

karya, NAIP-LI was highly enriched in neuronal processesthat formed a fibrous network (Fig. 5B).

Mesencephalon

In the substantia nigra, the majority of NAIP-LI neu-rons were located in the substantia nigra pars compacta.NAIP-LI was also present within the dendrites of theseneurons, which extend ventrally into the substantia nigrapars reticulata (Fig. 5C). A few NAIP-LI neurons wereobserved in the substantia nigra pars reticulata and parslateralis (Figs. 5C, 8A). Double labelling revealed that themajority of neurons that displayed NAIP-LI in theseregions were dopaminergic (Fig. 8B,C). The most intenselylabelled neurons in the midbrain were located in theoculomotor nucleus. Strongly labelled fibers were alsoapparent in the oculomotor nerve root (Figs. 9A, 11A).High levels of NAIP-LI were detected in neurons of theEdinger-Westphal nucleus and the trochlear nuclei (Fig.9A, Table 1). Both the parvocellular and the magnocellularparts of the red nucleus displayed intense NAIP-LI (Fig.9A). In the pontine nucleus, numerous small, round peri-karya were observed (Fig. 10A).

Met- and myelencephalon

Brainstem. The majority of cranial nerve nuclei in thebrainstem displayed strong NAIP-LI. Intensely NAIP1

neurons were present in all of the major nuclei of thetrigeminal nerve. These included the spinal, mesence-phalic, principal sensory, and motor nuclei of the trigemi-nal nerve (Figs. 9B,C, 10B). The strongest labelling wasdisplayed by medium-sized neurons in the mesencephalictrigeminal nucleus (Fig. 10B). Intense NAIP-LI was alsodetected in the vestibular nucleus, the cochlear nucleus,and nerve fibers of the vestibulocochlear nerve (Fig. 9B,D).Other cranial nerve nuclei that contained prominent label-ling included the facial nucleus, the nucleus of the solitarytract, the motor nucleus of vagus, and the hypoglossalnucleus (Fig. 9C,D, Table 1). Double labelling of the cranialnerve nuclei for NAIP and ChAT revealed that, in motornuclei, such as the oculomotor nucleus, the majority ofNAIP1 neurons were also immunoreactive for ChAT (Fig.11B,C). Neurons located within brainstem relay nuclei,such as the gracile nucleus, cuneate nucleus, gigantocellu-lar nucleus, and locus coeruleus, were also strongly stainedwith the NAIP antibody (Figs. 9D, 10B,C). In addition,

Fig. 8. NAIP-LI in the substantia nigra and double labelling ofnigral neurons for NAIP and tyrosine hydroxylase (TH). NAIP-LI wasdisplayed chiefly by neurons in the SNC (A). Double labelling of SNCneurons for NAIP (B) and TH (C) show the high cellular overlap ofthese twomarkers (arrowheads). Scale bars5 500 µm inA, 30 µm inC.

Fig. 9. NAIP-LI in cranial nerve and relay nuclei of the brainstem.Intense NAIP-LI was detected in several cranial nerve nuclei, such asthe oculomotor (3) and Edinger-Westphal (EW; A) nuclei, the trigemi-nal (DMSP5, SP50) and cochlear (VC; B) nuclei, the facial nucleus(7M, 7L;C), and the vagus (10) and hypoglossal (12) nuclei (D). Stronglabelling of the nerve roots extending ventrally from the oculomotor(3nr) and cochlear (8vn) nerve is apparent (A,B). NAIP1 neurons werealso observed in brainstem relay nuclei, such as the red nucleus (RMC,RPC; A), as well as the gracile (Gr) and cuneate (Cu) nuclei (D). Scalebar 5 400 µm.

Fig. 10. NAIP-LI in relay and reticular nuclei of the brainstem andcerebellum. A: NAIP-LI was observed in a large number of neurons inthe pontine nucleus (Pn) and the rostral periolivary area (RPO). B:The locus coeruleus (LC) as well as the three major nuclei of thetrigeminal nerve, i.e., the mesencephalic (Me5), principal sensory(Pr5), and motor (Mo5) nuclei, also displayed intense NAIP-LI. C:Neurons in the gigantocellular reticular nucleus were strongly la-belled (arrowheads indicate giant-sized neurons). D: Intense NAIP-LIwas also present in the vestibular nucleus (Ve) as well as the lateral(lat) and interposed (int) cerebellar nuclei (D). Scale bar 5 400 µm.

254 D.G. XU ET AL.

Figure 9

Figure 10

NAIP-LI was detected in the trochlear nucleus, nucleusambiguus, interpeduncular nucleus, inferior olivarynucleus, and lateral reticular nucleus (Table 1).Cerebellum. In the cerebellum, NAIP-LI perikarya

(round in shape) were observed in a single layer of the

cerebellar cortex (Fig. 12A). Double-immunofluorescencelabelling of these neurons with an antibody raised againstcalbindin D-28K, a marker protein for Purkinje cells,indicated that NAIP-LI was located in Purkinje cells (Fig.12B,C). NAIP-LI was not detected in any other layer of thecerebellar cortex. Cell bodies containing NAIP-LI werealso observed in deep cerebellar nuclei, such as the inter-posed and lateral nuclei (Fig. 10D).

Spinal cord

Intensely labelled perikarya that varied in size andmorphology were observed at various levels of the spinalcord, located primarily in the ventral horn (laminae VIIIand IX) and the intermediate zone (lamina VII; Figs. 13,14A,B). The most prominently labelled cells in the ventralhorn were very large neurons, presumably a-motor neu-rons (Fig. 14B). In keeping with this proposal, theseneurons were double labelled with the ChAT antibody (Fig.14C,D). Large perikarya were also observed in lamina VII,particularly in close proximity to the central canal. Clarke’scolumn, located in lamina VII, was strongly labelled withthe NAIP antibody. Small numbers of neurons were de-tected in laminae V and VI of the dorsal horn. A fewpositive neurons with small, round perikarya were alsoobserved in the dorsal horn (laminae I–IV; Fig. 14A).Finally, fiber staining was present within white matter ofthe spinal cord (Fig. 13).

DISCUSSION

We report here the use of an affinity-purified polyclonalantibody raised against NAIP to examine the distributionof NAIP-LI in the CNS. Immunohistochemical detection ofNAIP revealed that this antiapoptotic protein is presentwithin neurons located in a wide variety of structures.NAIP-LI was not detected in nonneuronal cell types likethose associated with the cerebrovasculature or glia, sug-gesting that NAIP is expressed selectively by neurons inthe CNS.In the forebrain, low levels of NAIP-LI were observed in

the cortex and the hippocampus. Within the cortex,NAIP-LI was observed in both superficial and deep layers.In addition to cell body labelling, NAIP-LI was clearlyvisible in apical dendrites of pyramidal neurons, suggest-ing that this antiapoptotic protein is actively transported.More extensive studies will be required both to confirmsuch transport and to establish the relative number andprecise nature of neurons that display NAIP-LI in thecerebral cortex. NAIP-LI was also widely distributed in thehippocampus, where it was detected within pyramidalneurons of the CA1–CA3 subfields, interneurons, andgranule cells of the dentate gyrus. NAIP-LI, as seen in thecortex, was localized in both the cell bodies and thedendrites of pyramidal neurons. The intensity of NAIPstaining appeared to be similar in CA1 and CA3, neuronssuggesting that NAIP is not responsible for the well-characterized differences in the vulnerability of theseneuronal populations to ischemic damage (Francis andPulsinelli, 1982). Within the thalamus, moderate levels ofNAIP-LI were observed in a large number of neuronslocated in ventral and lateral regions of this structure.Although NAIP-LI was also present in anterior, intralami-nar, and medial thalamic nuclei, fewer immunoreactiveneurons were detected in these areas. NAIP-LI was en-

Fig. 11. NAIP-LI in the oculomotor nucleus (3) and double-labelling immunohistofluorescence of oculomotor neurons for NAIPand ChAT. A: Example of NAIP-LI in the oculomotor nerve (3nr)showing cell body and axonal labelling. B: Immunohistofluorescencelabelling of NAIP in the oculomotor nucleus. C: Immunohistofluores-cence labelling for ChAT in the same region of the oculomotor nucleusshown in B. Arrowheads indicate double-labelled neurons, and arrowsshow examples of NAIP-immunoreactive neurons that do not containChAT. Scale bars 5 300 µm inA, 30 µm in C (also applies to B).

256 D.G. XU ET AL.

riched within fibers forming a dense network in ventrolat-eral aspects of the thalamus, further suggesting that NAIPis transported from the cell body to dendrites and axons.In contrast to the cortex, hippocampus, and thalamus,

NAIP-LI in the striatum and habenula was restricted to asmall subpopulation of neurons. In the striatum, double-labelling studies revealed that NAIP-LI was expressedexclusively in cholinergic interneurons, which account for1% of the total neuronal population in this structure(Semba and Fibiger, 1989). It is tempting to speculate thatthe high levels of NAIP-LI observed in these neurons mayaccount in part for their resistance to excitotoxic/ischemicinjury (Francis and Pulsinelli, 1982). Although NAIP-LIwas invariably present within striatal cholinergic neu-rons, NAIP-LI in the basal forebrain was rarely observedin cholinergic neurons. This finding may have relevance tothe observation that, within the basal forebrain, noncholin-ergic cells are more resistant than cholinergic neurons toretrograde injury produced by axotomy (Peterson et al.,1987).In addition to the cortex, striatum, and thalamus,

several other structures implicated in sensory and motorfunction were found to contain NAIP1 neurons. Theseincluded the globus pallidus, substantia nigra pars com-pacta, entopeduncular nucleus, pontine nuclei, lateralreticular nucleus, cerebellum, and spinal cord. The inten-sity of NAIP-LI in neurons of these structures suggeststhat this antiapoptotic proteinmay play a role inmaintain-ing their survival. Consistent with this proposal arereports of neuronal degeneration in the thalamus (ventraland lateral nuclei), globus pallidus, substantia nigra,pontine nuclei, cerebellum, and spinal cord of severe casesof SMA (Steiman et al., 1980; Towfighi et al., 1985;Murayama et al., 1991). The concordance between thedistribution of NAIP-LI and neuronal damage in SMAstrongly suggests that loss of NAIP contributes to pathol-ogy observed in this neurodegenerative disorder.In cranial nerve nuclei, high levels of NAIP-LI were

present in bothmotor and sensory neurons. ChAT immuno-histochemistry was utilized to assist identification of mo-tor neuron populations within the midbrain and brain-stem. Double-labelling studies revealed that NAIP-LI wasfrequently located in motor (ChAT1) neurons of the oculo-motor, trochlear, motor trigeminal, and facial nuclei; in themotor nucleus of vagus; and in the hypoglossal nucleus.These studies also demonstrated that NAIP-LI was pres-ent within sensory (ChAT2) neurons of cranial nuclei,which contain both motor and sensory neurons, such asthe facial nucleus (data not shown). Finally, intenseNAIP-LI was found in nuclei associated with sensoryfunction, i.e., Clarke’s column and gracile and cuneatenuclei. In further support of a central role for NAIPdepletion in SMA, all of these structures display damagein SMA (Steiman et al., 1980; Towfighi et al., 1985).

Fig. 12. NAIP-LI in the cerebellar cortex. A: NAIP1 somata withan orderly monolayer alignment were detected in the cerebellarcortex. NAIP-LI was not observed in any other layers. B: Immunohis-tofluorescence labelling of NAIP in the cerebellar cortex. C: Immuno-histofluorescence labelling of calcium-binding protein (CaBP; 28 kD)showing Purkinje cells (P) in the same region of the cerebellum shownin B. Note the perfect overlap between neurons immunoreactive forNAIP (B) and CaBP (C). G, granule cell layer; M, stratum moleculare.Scale bars 5 300 µm inA, 30 µm in C (also applies to B).

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Figure 13

Figure 14

In summary, the present study demonstrates thatNAIP-LI is distributed widely in the rat CNS. Althoughlow-to-moderate levels of NAIP-LI were observed in thecortex and hippocampus, intense NAIP-LI was detectedwithin a wide variety of subcortical brain regions. Theseincluded the thalamus (ventral and lateral nuclei), basalganglia (striatum, globus pallidum, endopeduncularnucleus, red nucleus, and substantia nigra), cranial nervenuclei, brainstem relay nuclei (cuneate, gracile, pontine,lateral reticular, interpeduncular, olivary nucleus, andlocus coeruleus), cerebellum, and spinal cord. Except forthe striatum, all of these regions displayed neuropathologyin severe cases of SMA. The fact that NAIP-LI in thestriatum is located exclusively in cholinergic interneurons,which account for only 1% of the striatal neuron popula-tion, may account for the failure of previous studies toobserve degenerative changes in this structure (Towfighiet al., 1985; Murayama et al., 1991).Neuronal abnormalities in SMAconsist of morphological

features, such as chromatolysis, which are consistent withthe process of apoptosis (Murayama et al., 1991). Becauseapoptosis plays an important role in neurodevelopment,this observation has led to the suggestion that a geneticdefect in a neuronal apoptotic pathway may be responsiblefor neuronal depletion in SMA (Sarnat, 1983). In supportof this hypothesis, acute SMA is associated with deletion ofa novel gene that encodes an inhibitor of apoptosis termedNAIP (Roy et al., 1995; Liston et al., 1996). A pivotal rolefor NAIP deletion in SMA is strongly suggested by ourobservation of a close anatomical correspondence betweenthe regional distribution of neuropathology in SMA andNAIP-LI. Determining themolecularmechanisms bywhichNAIP prevents apoptosis, therefore, may not only furtherour understanding of the development of this neurodegen-erative disorder, but it may also lead to novel therapeuticapproaches.

ACKNOWLEDGMENTS

We thank W. Tetzlaff and T. Crawford for helpful com-ments.

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Fig. 13. NAIP-LI in the spinal cord. Numerous NAIP1 perikaryathat varied in size and morphology were observed at the cervical (C5;A), thoracic (T2, T6; B,C), and sacral (S; D) levels of the spinal cord.Intensely labelled neurons are located predominantly in the anteriorpart of the spinal cord. Note that NAIP-LI is present within neuronalprocesses in both gray and white matter. Scale bar 5 400 µm.

Fig. 14. Distribution of NAIP-LI in the dorsal and ventral horns ofthe spinal cord at thoracic level 2. NAIP1 neurons are concentratedwithin the ventral horn (laminae VII–IX) and Clarke’s column (CC;lamina VII). Themajority of NAIP1 perikarya in the ventral horn werelarge and triangular (B, arrowheads). NAIP1 perikarya, although theywere fewer in number and smaller in size, were also seen in laminae Vand VI. A: Laminae I–IV contained few immunoreactive neurons. B:Immunohistofluorescence labelling for NAIP in the ventral horn of thespinal cord. C,D: Immunohistofluorescence labelling for ChAT (D) insame region of the ventral horn of the spinal cord shown in C.Arrowheads in C and D indicate double-labelled neurons. Scale bars 5300 µm inA (also applies to B), 30 µm in D (also applies to C).

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